ABSTRACT Accurate and efficient DNA replication is not merely a necessity for genome duplication, but constitutes an intrinsic barrier against genome instability. The ability to rapidly copy large genomes relies heavily on a sufficiently high number of functional replication complexes. Therefore, chromosomal regions that are devoid of replication origins or that exhibit structural impediments often display increased fragility. Minichromosome maintenance protein 10 (Mcm10) is an integral part of the replication machinery and has been identified as a potent protector against DNA breakage. This essential protein is required during replication initiation and elongation. Mcm10 facilitates DNA unwinding ? likely through its ability to bind single-stranded DNA ? and serves as a scaffold that is directly connected to and migrates with the replicative helicase. Mcm10 interacts with a number of replication and repair factors, including DNA polymerase (pol)-?/primase, the homotrimeric replication clamp, proliferating cell nuclear antigen (PCNA), the heterotrimeric checkpoint clamp 9-1-1 (Rad9/Rad1/Hus1), and the RecQ-like helicase RecQL4. Over the past decade my laboratory has revealed crucial aspects of Mcm10 biology, mostly by studying the protein in budding yeast. To explore the role of Mcm10 in human cells, we have generated heterozygous cell lines and found that MCM10 is haploinsufficient, contrary to the situation in yeast and mouse. Haploinsufficiency of MCM10 is particularly pronounced in oncogenically-transformed cells, resulting in a slow-growth phenotype that becomes increasingly severe with extended culture. Concomitantly, the cells exhibit progressive telomere shortening, despite expressing active telomerase. Based on these observations we hypothesize that Mcm10 is limiting for origin activation and telomere replication in humans. The overall goal of this proposal is to investigate the reason for MCM10's haploinsufficiency, its requirement in different cell-types and its exact role in telomere maintenance. The significance of this objective is further underscored by the observation that a loss-of-function mutation in MCM10 that inactivates one of the two parental alleles in the human germline causes the depletion of mature natural killer (NK) cells, leading to an inborn NK cell deficiency. The three Specific Aims of this proposal present a mechanistic dissection of MCM10 regulation in human cells. 1. Elucidate Mcm10's role in telomere maintenance 2. Determine the requirement for Mcm10 in transformed and non-transformed cells 3. Delineate the molecular defect of MCM10 mutations that cause NK cell deficiency